This invention relates to novel lysergic acid diethylamide derivatives and to the use of these derivatives in producing anti-lysergic diethylamide antibodies and to the use of these antibodies as reagents in improved immunoassays for lysergic acid diethylamide and its metabolites in biological fluid samples.
(+)-Lysergic acid diethylamide, 9,10-didehydro-N,N-diethyl-6-methylergoline-8xcex2-carboxamide, known as LSD, is a hallucinogen which acts on the central nervous system and alters sensory perception. A concentration of from 20 to 80 xcexcg of LSD is sufficient to induce hallucination (Nelson, C. and Foltz, R. Anal. Chem, 64, 1578-1585, 1992). The use of LSD has been and continues to be a problem for drug and law agencies around the world. Current methods used to determine the levels of LSD and its metabolites, for example N-desmethyl LSD (nor-LSD), in biological fluids such as serum and urine include fluorescence spectroscopy, GC/tandem MS, HPLC and radioimmunoassay (RIA). Fluorescence spectroscopy, however, exhibits nonspecific interference and does not distinguish between LSD, its metabolites, and other ergot alkaloids. HPLC results in lower nonspecific interference with antibody binding. However this method is time consuming and not suitable for routine screening of large numbers of samples. Immunoassays offer many advantages over the aforementioned analytical methods, including rapid drug screening and most importantly, high sensitivity.
In immunoassays for drugs such as LSD, a biological fluid sample suspected of containing said drug or its metabolites is contacted with antibodies in the presence of a labeled LSD derivative (label). To the extent that the drug or its metabolites are present in the sample, there will be competition for binding to the antibodies and the amount of the labeled derivative that remains bound will be reduced in proportion to the degree of competition with the drug or its metabolites in the sample.
A fluorescence immunoassay for LSD has been described (B. Law et al., Anal. Proc. 20, 606, 1983). The immunoconjugate that is used to generate antibodies is prepared out of the carboxyl residue of lysergic acid (see e.g., H. Van Vunakis Proc. Nat. Acad. Sci. 68, 1483-1487, 1971) and antibodies derived therefrom would be expected to have high cross-reactivity with a number of ergot alkaloids. The tracer used in this method is produced by conjugating fluoresceinamine to the acid residue of the molecule. The sensitivity of the assay system is in the range of 5-40 ng/mL of LSD. Although this method has the advantage of being rapid, it has a poorer limit of detection than the RIA.
A radioimmunoassay for LSD is described wherein an LSD immunoconjugate is prepared out of the carboxy residue of lysergic acid (see e.g., H. Van Vunakis Proc. Nat. Acad. Sci. 68, 1483-1487, 1971). However, the antibodies derived from the immunoconjugate are not highly specific for LSD. Several ergot alkaloids such as ergosine, ergonovine, ergotamine and methysergide (also known as methylergonovine, Sandoz) are shown to compete for binding to the LSD antibodies, resulting in undesirable false positive results.
Another radioimmunoassay for LSD in serum and urine samples is described (see W. A. Ratcliffe, Clin. Chem. 23(2), 169-174, 1977), wherein the immunoconjugate is prepared via a Mannich type reaction (J. March, Advanced Org. Chem. 4th ed., 900 (1992)), by condensation of LSD and bovine serum albumin (BSA) in the presence of formaldehyde (Tauton-Rigby, Science, 181, 165, 1973). The immunoconjugate produced generates LSD antibodies having low cross-reactivity to a number of ergot alkaloids. The linkage formed in the condensation is derived out of the indole group of the LSD molecule. 
This linkage is similar to an acetal and imidazoline (herein termed an xe2x80x9caminoketalxe2x80x9d) which serve as the nitrogen protecting groups of indole. As these groups are each susceptible to hydrolysis in an acidic medium, this linkage can also be readily cleaved by hydrolysis in an acidic medium (see e.g., A. J. Stern and J. S. Swenton, J. Org. Chem. 54, 2953 (1989); D. A. Evans, et al., J. Am. Chem. Soc., 103, 5813 (1981); and A. Giannis, et al., Tetrahedron, 44, 7177 (1988)). N,Nxe2x80x2-diisopropylidenephenylalanylleucine bearing the xe2x80x9caminoketalxe2x80x9d linkage is cleaved simply by heating the material in 10% aqueous solution at 60xc2x0 C. at neutral pH. (see P. M. Hardy, D. J. Samworth, J. Chem. Soc., Perkin I, 1954 (1977)). Therefore, a method resulting in this unstable linkage is not suitable for making LSD derivatives to be used in preparing haptens and reagents bearing a stable linkage for immunoassay.
Therefore, a non-radioisotopic immunoassay method to rapidly detect LSD and its metabolites, especially in urine samples, is highly desirable. It is also desirable to have an LSD derivative bearing a stable noncleavable linkage from which stable haptens and immunogens can be generated. Furthermore, antibodies used to detect LSD and its metabolites should be highly specific and should not cross-react with other ergot alkaloids.
The present invention provides hapten derivatives that are useful for the preparation of antigenic, antibody and labelled reagents having superior performance characteristics for use in immunoassays for the detection of lysergic acid diethylamide (LSD) and its metabolites. In one embodiment of the present invention, the LSD nucleus is derivatized out of the indole nitrogen to form an aminoalkyl haptenic derivative. In another embodiment, the derivatives are produced out of the piperidine nitrogen of the nor-LSD molecule. The preferred derivatization is through alkylation forming a functional group which can be further modified for linking to an appropriate linking, antigenic or labelling group to provide stable reagents for the immunoassay.